Identification of a Novel Vasodilatory Octapeptide from the Skin Secretion

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Article Identiﬁcation of a Novel Vasodilatory Octapeptide from the Skin Secretion of the African Hyperoliid Frog, Kassina senegalensis

Qiang Du 1,†, Hui Wang 1,*,†, Chengbang Ma 2, Yue Wu 2, Xinping Xi 2,*, Mei Zhou 2 ID , Tianbao Chen 2 ID , Chris Shaw 2 and Lei Wang 2

1 School of Pharmacy, China Medical University, Shenyang 110001, Liaoning, China; [email protected] 2 Natural Drug Discovery Group, School of Pharmacy, Queen’s University, Belfast BT9 7BL, Northern Ireland, UK; [email protected] (C.M.); [email protected] (Y.W.); [email protected] (M.Z.); [email protected] (T.C.); [email protected] (C.S.); [email protected] (L.W.) * Correspondence: [email protected] (H.W.); [email protected] (X.X.); Tel.: +86-24-2325-6666 (H.W.); +44-28-9097-2200 (X.X.); Fax: +86-2325-5471 (H.W.); +44-28-9094-7794 (X.X.) † These authors contributed equally to this work.

Received: 5 July 2017; Accepted: 19 July 2017; Published: 19 July 2017

Abstract: The defensive skin secretions of amphibians continue to be an excellent source of novel biologically-active peptides. Here we report the identiﬁcation and pharmacological activity of a novel C-terminally amided myotropic octapeptide from the skin secretion of the African hyperoliid frog, Kassina senegalensis. The 8-amino acid peptide has the following primary structure: WMSLGWSL-amide and has a molecular mass of 978 Da. The primary structure and organisation of the biosynthetic precursor of WL-8 amide was successfully deduced from cloned skin secretion-derived cDNA. The open-reading frame encoded a single copy of WL-8, located at the C-terminus. Synthetic WL-8 amide was found to cause relaxation of rat tail artery smooth muscle with an EC50 of 25.98 nM. This peptide is unique in terms of its primary structure and is unlike any other peptide previously isolated from an amphibian source which has been archived in the NCBI database. WL-8 amide thus represents the prototype of a novel family of myotropic peptide from amphibian defensive skin secretions.

Keywords: amphibian; skin secretion; peptide; smooth muscle

1. Introduction The skin secretions of anurans (frogs and toads) have long been established as an excellent source of structurally-unique peptides which can be used as lead compounds in research into novel therapeutic agents [1,2]. These peptides have been honed through natural selection and physiological adaptations to aid the survival of the organism over millions of years [3]. A wide range of pharmacologically-active peptides are present in skin secretions. These peptides may exert some biological effect or even be highly toxic to would-be predators [3]. An example which demonstrates the physiological effects these peptides can have on predators has been reported in a study of the garter snake. The defensive secretions produced by Xenopus can cause oral dyskinesias in the snake, such that it cannot physically close its mouth [4]. This effect, if reproduced on similar predators in Nature, would certainly permit the frog to escape. Many of the peptides isolated from amphibians exert their pharmacological functions by acting on speciﬁc targets within the body, for instance at G protein-linked receptors. This targeting ability of peptides makes them very interesting molecules in the search for potential drug candidates. The pharmacologically-active peptides found in the skin secretions of amphibians are often found to

Molecules 2017, 22, 1215; doi:10.3390/molecules22071215 www.mdpi.com/journal/molecules Molecules 2017, 22, 1215 2 of 9 Molecules 2017, 22, 1215 2 of 8 befunctions analogues in humans of the naturally-occurring and in other vertebrates. regulatory Some are or neuropeptideseven identical to which their endogenous carry out physiological mammalian functionscounterparts in humans such as and thyrotropin-releasing in other vertebrates. hormon Some aree (TRH) even identical[5] and the to theircanonical endogenous bradykinin mammalian peptide, counterpartsbut most are suchnot identical as thyrotropin-releasing though they do hormonehave highly-conserved, (TRH) [5] and thebiologically-active canonical bradykinin regions peptide, within buttheir most structures are not [6]. identical This latter though group they includes do have highly-conserved,bombesin, which is biologically-active an analogue of gastrin-releasing regions within theirpeptide structures in humans [6]. and This also latter the group caeruleins, includes which bombesin, are similar which to gastrin is an analogue and cholecystokinin of gastrin-releasing found in peptidehumans in [7]. humans and also the caeruleins, which are similar to gastrin and cholecystokinin found in humansFrog [7 ].skin secretion is a complex mixture of proteins and peptides which have a variety of pharmacologicalFrog skin secretion effects such is a as complex neurotoxicity mixture and of myotoxicity. proteins and Many peptides proteins which and havepolypeptides a variety have of pharmacologicalbeen isolated from effects amphibian such asskin neurotoxicity which exhibit and a myotoxicity.contractile effect Many on proteinsrat bladder and smooth polypeptides muscle. haveHowever, been the isolated effects fromof low amphibian molecular-weight skin which peptides exhibit on such a contractile smooth muscles effect onare ratnot bladderfully understood. smooth muscle.Here, However, the discovery the effects of a prototype of low molecular-weight low molecular mass peptides peptide on suchfrom smooththe skin muscles secretion are of notthe fullyAfrican understood. hyperoliid frog, Kassina senegalensis, is reported. This peptide represents an entirely novel structuralHere, class the discovery of amphibian of a prototypeskin peptide low and molecular was found mass to display peptide a fromunique the relaxation skin secretion of rat ofartery the Africansmooth hyperoliidmuscle. The frog, peptideKassina was senegalensis named, WL-8, is reported. amide (978 This Da) peptide in accordance represents with an its entirely chain length novel structuraland first and class last of amino amphibian acid skinresidues. peptide We andfurther was de foundscribe to the display structure a unique of its relaxationbiosynthetic of ratprecursor artery smoothidentified muscle. through The peptidemolecular was cloning named, of WL-8 its amideencoding (978 Da)cDNA in accordancefrom a skin with secretion-derived its chain length andcDNA ﬁrst library. and last amino acid residues. We further describe the structure of its biosynthetic precursor identiﬁed through molecular cloning of its encoding cDNA from a skin secretion-derived cDNA library. 2. Results 2. Results Each specimen of K. senegalensis yielded on average 30–35 mg dry weight of skin secretion K. senegalensis followingEach manual specimen stimulation. of Five mg ofyielded lyophilised on average skin secretion 30–35 was mg subjec dry weightted to reverse-phase of skin secretion high followingperformance manual liquid stimulation. chromatography Five mg(RP-HPLC) of lyophilised fractionation skin secretion that produced was subjected a complex to reverse-phasechromatogram high(Figure performance 1). liquid chromatography (RP-HPLC) fractionation that produced a complex chromatogram (Figure1).

1484

1081

678 WL-8 amide Absorbance [mA] Absorbance

275

-128

83:30 91:58 100:25 108:53 117:20 125:48 Time [mm:ss]

FigureFigure 1.1. Region of reverse-phase high high performance performance liqu liquidid chromatography chromatography (RP-HPLC) (RP-HPLC) chromatogram chromatogram of oflyophilised lyophilised KassinaKassina senegalensis senegalensis skinskin secretion. secretion. The Theretention retention time timeof the of WL-8 the WL-8 peptide peptide is 97 min is 97 (32.3% min (32.3%acetonitrile) acetonitrile) and indicated and indicated by an arrow. by an arrow.

Chromatographic fractions were screened using the smooth muscle bioassay. The single peptide in fraction #97, which was eluted around 32% acetonitrile, exhibiting myotropic activity, was

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Chromatographic fractions were screened using the smooth muscle bioassay. The single peptide Molecules 2017, 22, 1215 3 of 8 in fraction #97, which was eluted around 32% acetonitrile, exhibiting myotropic activity, was sequenced sequencedunambiguously unambiguously by LC/MS/MS by LC/MS/MS fragmentation fragmentation (Figure2 (Figure). The amino2). The acidamino sequence acid sequence of WL-8 of wasWL- 8determined was determined as: Trp-Met-Ser-Leu-Gly-Trp-Ser-Leu-amide as: Trp-Met-Ser-Leu-Gly-Trp-Ser-Leu-amide (WMSLGWSL). (WMSLGWSL). The WL-8 amide-encoding precursor precursor cDNA cDNA was was successfully cloned cloned from from the skin secretion librarylibrary using using the RACE protocol described. The The open-reading open-reading frame frame consisted consisted of of 76 76 amino acid residuesresidues (Figure 33).). TheThe maturemature peptidepeptide waswas ﬂankedflanked N-terminally by identical propeptide convertaseconvertase processing sitessites probablyprobably involving involving a proteasea protease with with Lys-Arg Lys-Arg cleavage cleavage speciﬁcity. specificity. The last The glycyl last residueglycyl residuein the open-reading in the open-reading frame frame is appropriately is appropriately positioned positioned to act to as act an as amide an amide donor donor for for generation generation of ofthe the C-terminal C-terminal leucinamide leucinamide residue residue present present on on the the mature mature peptide. peptide. TheThe nucleotidenucleotide sequence of the WL-8 precursorprecursor has has been been deposited deposited in the in European the European Molecular Molecular Biology LaboratoryBiology Laboratory (EMBL) Nucleotide (EMBL) NucleotideSequence Database Sequence under Database the accession under the code accession LT634112. code LT634112. InIn order order to toproduce produce a large a large quantity quantity of replic ofate replicate of the natural of the peptide, natural it peptide,was chemically- it was synthesisedchemically-synthesised by solid phase by solidFmoc phase chemistry Fmoc and chemistry the resultant and the peptide resultant was peptide lyophilized. was lyophilized. The resulting The productresulting was product of an wasapparent of an apparenthigh degree high of degreepurity ofas puritydetermined as determined by MALDI-TOF by MALDI-TOF MS. MS. This synthetic replicate of WL-8 amide produced a dose-dependent relaxation of rat arterial smoothsmooth muscle though molar potencies were found to vary with preparations (Figure 44).). AA fullfull dosedose responseresponse curve was constructed. Repeat Repeat experiment experimentss using using the the pure pure synthetic synthetic replicate replicate in in several different smooth muscle muscle preparations preparations showed showed that that it it was was devoid devoid of of myorelaxant myorelaxant activity activity in in these. these. InIn a a further further seriesseries of of experiments, experiments, a a dose-dependent dose-dependent relaxation relaxation was was observed observed for WL-8 WL-8 peptide in the the −11 −5 −7 rangerange of of 10 10−11–10–10−5 M Mwith with a maximal a maximal effective effective concentration concentration of 10 of−7 10 M. Furthermore,M. Furthermore, the peptide the peptide was was found to have an EC of 25.98 nM suggesting that this peptide is a very potent vasorelaxant. found to have an EC50 of 25.9850 nM suggesting that this peptide is a very potent vasorelaxant.

L SWG L S MW WM SLG W S L 979 #36-143 RT: 0.45-2.09 AV: 108 NL: 1.22E1 T: ITMS + p ESI E Full ms2 [email protected] [105.00-1000.00] b6 761.16 100

85 2+ [M-NH3] 80 480.60

Relative Abundance 40 y2 35 218.00 30

2 25 y -NH3 200.88 c2 20 4 336.04 b y6-NH 15 518.16 3 7 4 5 626.16 831.32 b 173.04 2 y -NH3 b 10 b 6 7 848.08 444.00 y 743.08 b -H2O 1 3 575.28 5 y 317.92 b y5 956.24 0 200 300 400 500 600 700 800 900 1000 m/z Figure 2. Annotated MS/MS fragmentation spectrum of natural WL-8 amide. Figure 2. Annotated MS/MS fragmentation spectrum of natural WL-8 amide.

Molecules 2017, 22, 1215 4 of 9 Molecules 2017, 22, 1215 4 of 8

Molecules 2017, 22, 1215 4 of 8 Signal Pept ide ORF WL-8-KS

Signal Pept ide ORF WL-8-KS

WL-8-KS 330 bp

WL-8-KS 330 bp M L S L K K S M L L L F F L G M V · 1 ATGCTGTCTT TGAAGAAATC CATGTTGCTG CTTTTCTTCC TTGGGATGGT TACGACAGAA ACTTCTTTAG GTACAACGAC GAAAAGAAGG AACCCTACCA M · SL SS SL LK AK QS S M RL RL E L AF EF AL EG DM KV ·R · 151 ATGCTGTCTTCTCGTCTTCC TGAAGAAATC CTAGCTCAAA CATGTTGCTG GCAGGAGAGA CTTTTCTTCC AGCTGAGGCT TTGGGATGGT GAAGATAAGA TACGACAGAAGAGCAGAAGG ACTTCTTTAG GATCGAGTTT GTACAACGAC CGTCCTCTCT GAAAAGAAGG TCGACTCCGA AACCCTACCA CTTCTATTCT · ·S AS DS E L EA GQ ES DR KR RE A A DE EA E E GD EK DR · 51101 CTCGTCTTCCGAGCTGATGA CTAGCT AGAGGGAGAACAAA GCAGGAGAGA GATAAGAGAG AGCTGAGGCT CTGATGAAGA GAAGATAAGA GGGAGAAGAT GAGCAGAAGGCTCGACTACT GATCGA TCTCCCTCTTGTTT CGTCCTCTCT CTATTCTCTC TCGACTCCGA GACTACTTCT CTTCTATTCT CCCTCTTCTA · KA RD AE D E EG EE G D EK DR KA RD KE RE W G ME SD L · 101 151 GAGCTGATGAAAGAGAGCTG AGAGGGAGAA ATGAAGAGGG GATAAGAGAG AGAAGATAAG CTGATGAAGA AGAAAGAGAT GGGAGAAGAT GGATGTCTCT CTCGACTACTTTCTCTCGAC TCTCCCTCTT TACTTCTCCC CTATTCTCTC TCTTCTATTC GACTACTTCT TCTTTCTCTA CCCTCTTCTA CCTACAGAGA K · GR WA SD LE GE *G E D K R K R W M S L · 151 201 AAGAGAGCTGGGGCTGGTCT ATGAAGAGGG CTGGGCTGAA AGAAGATAAG CTCCCACCTC AGAAAGAGAT TGTGGAAGAC GGATGTCTCT AATTCATATC TTCTCTCGACCCCGACCAGA TACTTCTCCC GACCCGACTT TCTTCTATTC GAGGGTGGAG TCTTTCTCTA ACACCTTCTG CCTACAGAGA TTAAGTATAG 251 · ATCTGATTACG W S LACATTCATAA G * TTCAGATGTC TTAATAAAAA ATATATTTGA 201 GGGCTGGTCTTAGACTAATG CTGGGCTGAA TGTAAGTATT CTCCCACCTC AAGTCTACAG TGTGGAAGAC AATTATTTTT AATTCATATC TATATAAACT 301 CCCGACCAGAATAATCAAAA GACCCGACTT AAAAAAAAAA GAGGGTGGAG AAAAAAAAAA ACACCTTCTG TTAAGTATAG 251 ATCTGATTACTATTAGTTTT ACATTCATAA TTTTTTTTTT TTCAGATGTC TTTTTTTTTT TTAATAAAAA ATATATTTGA TAGACTAATG TGTAAGTATT AAGTCTACAG AATTATTTTT TATATAAACT 301 ATAATCAAAA AAAAAAAAAA AAAAAAAAAA TATTAGTTTT TTTTTTTTTT TTTTTTTTTT FigureFigure 3.3. NucleotideNucleotide and translatedtranslated open-readingopen-reading frameframe amino acid sequencesequence ofof full-lengthfull-length cDNAcDNA encoding a single copy of WL-8. The putative signal peptide (blue colour and single-underlined), the Figureencoding 3. Nucleotide a single copy and of translated WL-8. The open-reading putative signal fram peptidee amino (blue acid colour sequence and single-underlined), of full-length cDNA the mature encoding sequence (double-underlined) and the stop codon (asterisk) are indicated. encodingmature encoding a single copy sequence of WL-8. (double-underlined) The putative sign andal peptide the stop (blue codon colour (asterisk) and single-underlined), are indicated. the mature encoding sequence (double-underlined) and the stop codon (asterisk) are indicated.

Relaxation(g) Relaxation(g)

Figure 4. Dose–response curves of relaxation effects on phenylephrine stimulated rat tail artery smooth muscle preparation in the Bradykinin (▲) or WL-8 amide (■). Each point represents the mean FigureFigure 4. Dose–response curvescurves of of relaxation relaxation effects effects on phenylephrineon phenylephrine stimulated stimulated rat tail rat artery tail smoothartery and standard error. (4 repilicates, 3 individual experiments.) EC50 of Bradykinin: 1.29 nM; EC50 of WL- smoothmuscle muscle preparation preparation in the Bradykininin the Bradykinin ( ) or ( WL-8▲) or amideWL-8 amide ( ). Each (■). Each point point represents represents the meanthe mean and 8 amide: 25.98 nM. N andstandard standard error. error. (4 repilicates, (4 repilicates, 3 individual 3 individual experiments.) experiments.) EC EC50 of50 of Bradykinin: Bradykinin: 1.29 1.29 nM; nM; EC EC5050of of WL-8WL- amide: 25.98 nM. 3. Discussion8 amide: 25.98 nM.

3.3. Discussion DiscussionThe skin secretions of amphibians continue to be an excellent source for the discovery of novel peptides [8,9]. The pharmacological effects of a novel amidated octapeptide, named WL-8, which was TheThe skin skin secretions secretions of of amphibians amphibians continue continue to to be be an an excellent excellent source source for for the the discovery discovery of of novel novel isolated from the skin secretions of the African running frog, Kassina senegalensis, were determined peptidespeptides [8,9]. [8,9]. The The pharmacological pharmacological effects effects of of a a novel novel amidated amidated octapeptide, octapeptide, named named WL-8, WL-8, which which was was on smooth muscle preparations and the peptide was found to be a potent relaxant of vascular smooth isolatedisolated from from the the skin secretionssecretions ofof thethe African African running running frog, frog,Kassina Kassina senegalensis senegalensis, were, were determined determined on muscle. An online protein/peptide database search revealed that no identical or even similar peptide onsmooth smooth muscle muscle preparations preparations and and the the peptide peptide was was found found to to bebe aa potentpotent relaxant of vascular vascular smooth smooth sequence has yet been reported. Therefore, it was concluded that WL-8 is a novel biologically-active muscle.muscle. An An online online protein/peptide protein/peptide database database search search re revealedvealed that that no no identical identical or or even even similar similar peptide peptide peptide. This peptide has two tryptophan (W) residues in its primary structure which is a rare finding sequencesequence has has yet yet been been reported. reported. Therefore, Therefore, it it was was concluded concluded that that WL-8 WL-8 is is a a novel novel biologically-active biologically-active in such a small peptide, additionally, it possesses two leucine residues and a single methionine. Thus peptide.peptide. This This peptide peptide has has two two tryptophan tryptophan (W) (W) residues residues in in its its primary primary structure structure which which is is a a rare rare finding ﬁnding 5 of the 8 amino acids that constitute this novel peptide are hydrophobic. Following a range of smooth inin such such a asmall small peptide, peptide, additionally, additionally, it possesses it possesses two twoleucine leucine residues residues and a and single a singlemethionine. methionine. Thus muscle bioassays, the peptide was found to be a potent vasorelaxant (EC50 of 25.98 nM) in the rat tail 5Thus of the5 8 of amino the 8 aminoacids thatacids consti thattute constitute this novel this peptid novele peptideare hydrophobic. are hydrophobic. Following Following a range of a rangesmooth of artery as shown in Figure 4. muscle bioassays, the peptide was found to be a potent vasorelaxant (EC50 of 25.98 nM) in the rat tail As we used the pre-constricted rat tail artery smooth muscle by the stimulation of phenylephrine, artery as shown in Figure 4. the tissue could generate a low degree of spontaneous relaxant in the period time of equilibration. As we used the pre-constricted rat tail artery smooth muscle by the stimulation of phenylephrine, Therefore, we compared the vasorelaxant effect of WL-8 with bradykinin using the same tissue the tissue could generate a low degree of spontaneous relaxant in the period time of equilibration. Therefore, we compared the vasorelaxant effect of WL-8 with bradykinin using the same tissue

Molecules 2017, 22, 1215 5 of 9

smooth muscle bioassays, the peptide was found to be a potent vasorelaxant (EC50 of 25.98 nM) in the rat tail artery as shown in Figure4. As we used the pre-constricted rat tail artery smooth muscle by the stimulation of phenylephrine, the tissue could generate a low degree of spontaneous relaxant in the period time of equilibration. Therefore, we compared the vasorelaxant effect of WL-8 with bradykinin using the same tissue preparation and we can therefore confidently state that WL-8 peptide from Kassina senegalensis is a potent relaxant of arterial smooth muscle as it shown similar potency to bradykinin. This high degree of molar potency would suggest that it mediates its action via a specific molecular target, rather than through a generalised effect, for instance via membrane interaction. But the targeted receptors where WL-8 can specifically bind need to be further investigated. Bioinformatic analysis of the structure of WL-8 amide found that it is unlike any other peptide previously isolated from an amphibian source. When the peptide is entered into the NCBI BLAST database, the closest matches from an animal source are the 38-mer cytokines, interferon gamma-1 and -2 isolated from rainbow trout (Oncorhynchus mykiss). The peptide exhibits a high degree of identity with the N-terminal regions of these fish cytokines. It may be that this peptide mimics their action in promoting inflammatory responses but this aspect may constitute a further series of studies. As this peptide is not structurally similar to anything previously isolated from an amphibian source its presence in the defensive secretions of Kassina senegalensis is most intriguing; it is unclear at this stage what its normal physiological action may be. Bradykinin and structurally-related peptides are often significant components of amphibian defensive skin secretions and are found in representative species of the families, Ranidae, Hylidae and Bombinatoridae [6]. The actions of bradykinin are well known and diverse, such as vasodilation with subsequent induced hypotension, induction of pain and relaxation of a variety of smooth muscle types. However, the bradykinin and bradykinin-related peptides have not been found from the skin secretion of Kassina species yet (Uniport database, July 2017). In this study, the novel WL-8 peptide has similar myotropic effects to the bradykinin as it causes relaxation of smooth muscle; however, its structure is dissimilar to those of all the bradykinin like peptides previously discovered. Interestingly, we did not find any bradykinin or bradykinin related peptides from its skin secretion. It is speculated that different frogs evolved to produce specific-targeting peptides against the predators in their circumstances, of which WL-8 might induce similar or more potent effects than bradykinin in the bodies of the predators, and it eventually substituted for bradykinin in their defence strategy. Myotropic peptides are widely-distributed throughout the Animal Kingdom and play an important role in the tissues of animals [10]. Their effects are well known and diverse and include vasodilation with subsequent induced hypotension, induction of pain and relaxation of a variety of smooth muscle types. They exert their effects upon smooth muscle which is found in the gastrointestinal tract, the urinary tract and within the walls of blood vessels. There is therefore a potential to produce new therapeutic agents which will mediate some form of pharmacological effect at one of these sites, and so this peptide may have the potential to be used to treat diseases affecting at least one of these systems. The African hyperoliid frog, Kassina senegalensis, has proven to be a valuable source of novel peptides which have the potential to be developed into new therapeutic agents [11,12]. Of the peptides isolated from this frog so far, some of those which show promise as potential therapeutic agents include the kassorins which possess antibiotic activity [12], and kassinin, a tachykinin possessing neurotransmitter and hormonal activity in higher vertebrates [13]. These discoveries, together with the novel peptide WL-8, indicate the potential this species has as a source for structurally-unique lead compounds or those that may reveal novel drug targets. Molecules 2017, 22, 1215 6 of 9

4. Materials and Methods

4.1. Specimen Biodata and Harvesting of Skin Secretion Specimens of K. senegalensis (n = 12) were obtained from several sources. The frogs were adults (4–5 cm snout to vent length) and were kept for a period of 4 months prior to secretion harvesting. They were maintained in our purpose-designed amphibian facility at 18–25 ◦C under a 12 h/12 h light/dark cycle and fed multivitamin-loaded crickets three times per week. Defensive skin secretions were obtained from the dorsal skin surface by gentle transdermal electrical stimulation using the technique of Tyler et al. [14] under appropriate UK animal research licences. Brieﬂy, the moistened skin was stimulated by platinum electrodes (6 V DC; 4 ms pulse-width; 50 Hz) for two periods of 20 s duration following which the secretion was washed from the skin using deionised water, snap-frozen in liquid nitrogen and lyophilised. Lyophilisate was stored at −20 ◦C prior to analysis. Sampling of skin secretion was performed by Mei Zhou under UK Animal (Scientiﬁc Procedures) Act 1986, project licence PPL 2694, issued by the Department of Health, Social Services and Public Safety, Northern Ireland. Procedures had been vetted by the IACUC of Queen’s University Belfast, and approved on 1 March 2011.

4.2. Fractionation of Skin Secretion by Reverse-Phase HPLC A five milligram sample of lyophilised skin secretion from K. senegalensis was reconstituted in 0.05/99.5 (v/v) trifluoracetic acid (TFA)/water and clarified of particulates by centrifugation. The supernatant was subjected to fractionation by pumping directly onto an HPLC column (Jupiter C-5, 5 µm particle, 300 Å pore, 250 mm × 10 mm, Phenomenex, UK). The peptides were eluted using a gradient formed from 0.05/99.95 (v/v) TFA/water to 0.05/19.95/80.00 (v/v/v) TFA/water/acetonitrile in 240 min. A gradient reverse phase HPLC system was employed and fractions were collected automatically at 1 min intervals. Two 100 µL samples from each chromatographic fraction were removed, lyophilised and stored at −20 ◦C prior to analysis for smooth muscle and antimicrobial activities.

4.3. Rat Tail Artery Smooth Muscle Bioassay Male Wistar rats (250–300 g) were euthanised by carbon dioxide asphyxiation followed by cervical dislocation under appropriate UK animal research licences. The rats were placed dorsal surface down and the abdomen was opened by means of an incision along the mid ventral line and subcutaneous fat was carefully dissected. The exposed urinary bladder was removed from each rat, emptied of urine and placed in ice-cold Kreb’s solution (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3, 1.15 mM NaH2PO4, 2.5 mM CaCl2, 1.1 mM MgCl2 and 5.6 mM glucose), equilibrated with 95% O2, 5% CO2. A 2 mm width ring of rat tail artery was dissected under a dissection microscope and connected to two triangular hooks with a fine silk thread (0.2 mm diameter) with one end subsequently attached to a fixed pin and the other to a transducer in a 2 mL organ bath containing Kreb’s solution at 37 ◦C flowing at 2 mL/min with constant bubbling of 95% O2, 5% CO2. Muscle rings were pre-contracted with 10 µM phenylephrine and equilibrated for 1 h before experimental procedures were initiated. Following this, viable preparations were used to screen samples of reverse phase HPLC fractions of K. senegalensis skin secretion for myoactivity. Subsequently, once the novel active peptide had been identified, structurally characterised and chemically-synthesised, muscle strips were used to analyse dose–response relationships. WL-8 amide solutions, ranging in concentration from 10−11 to 10−5 M, were made in Kreb’s solution and were used to construct a dose–response curve. These were added to the pre-constricted rat tail artery smooth muscle rings, using 10 µM phenylephrine, in increasing concentrations with 5 min equilibration periods between each dose. Each concentration of WL-8 amide solution was applied to a minimum of six muscle strips. Changes in tension of the artery smooth muscle rings were recorded and amplified through pressure transducers connected to a PowerLab System Molecules 2017, 22, 1215 7 of 9

(AD Instruments Pty Ltd, Oxford, UK). Data were analysed to obtain the mean and standard error of responses by one-way ANOVA and dose–response curves were constructed using a “log(agonist) vs. response (three parameters), Least squares ﬁt” algorithm through Prism 6 software package provided.

4.4. Structural Characterisation Using MS/MS Fragmentation Sequencing The peptide in the reverse-phase HPLC fraction which was found to possess smooth muscle relaxing activity, was subjected to tandem MS/MS fragmentation sequencing using an LCQ-Fleet electrospray ion-trap mass spectrometer (Thermo Fisher Scientiﬁc, San Francisco, CA, USA). In detail, the sheath and auxiliary gas ﬂow rates were set to 20 and 5 arbitrary units, respectively. The spray voltage was set to 4.50 kV while the capillary temperature was set to 275 ◦C. The fragmentation mode was collision-induced dissociation (CID) and normalised collision energy (NCE) was set as 35.

4.5. Molecular Cloning of WL-8 Amide Biosynthetic Precursor—Encoding cDNA Five milligrams of lyophilised skin secretion were dissolved in 1 mL of cell lysis/mRNA protection buffer that was obtained from Invitrogen. Polyadenylated mRNA was isolated from this by using magnetic oligo-dT Dynabeads as described by the manufacturer (Invitrogen, Vilnius, Lithuania). The isolated mRNA was then subjected to 50 and 30-rapid amplification of cDNA ends (RACE) procedures to obtain full-length WL-8 amide precursor nucleic acid sequence data using a SMART-RACE kit (Clontech, Palo Alto, CA, USA) likewise as per manufacturer’s instructions. Briefly, the 30-RACE reactions employed a nested universal (NUP) primer (supplied with the kit) and a degenerate sense primer (S: 50-TGGATGWSIYTIGGITGG-30) that was complementary to the N-terminal amino acid sequence, WMSLGW-, of the novel peptide, WL-8 amide. The 30-RACE reactions were purified and cloned using a pGEM-T vector system (Promega Corporation, Southampton, UK) and sequenced using an ABI 3100 automated sequencer. The sequence data obtained from the 30-RACE product was used to design a specific antisense primer (AS: 50-GTCTTCCACAGAGGTGGGAGT-30) to a defined conserved site within the 30 non-translated region of the WL-8 amide encoding transcripts. 50-RACE was carried out using these primers in conjunction with the NUP primer and resultant products were purified, cloned and sequenced.

4.6. Chemical Synthesis of WL-8 Amide WL-8 amide was synthesised automatically using the solid-phase method (MBHA Rink Amide- resin, 0.6 mmol/g, Merck Millipore, Nottingham, UK) and 8 standard fluorenylmethoxycarbonyl (Fmoc) chemistry (double couplings with 8 equivalents of Fmoc-amino acid derivatives) using a PS3 automated solid phase peptide synthesiser (Protein Technologies, Tucson, AZ, USA). Couplings were performed using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (Calbiochem-Novabiochem AG, Läufelfingen, Switzerland). Solid phase peptide synthesis begins with the covalent attachment (via its carboxyl group) of the C-terminal amino acid to an insoluble particle such as a polystyrene resin. The N-terminal end of the bound peptide undergoes coupling with carboxyl-activated amino-protected amino acids which results in the addition of residues to the expanding peptide, one amino acid at a time. Each step of this process includes a deprotection reaction and a coupling reaction. Upon completion of the peptide, it is chemically-cleaved from the resin. The peptide was purified by reverse-phase HPLC and its purity and molecular mass were confirmed using MALDI-TOF mass spectrometry.

Acknowledgments: This work was supported by Science Foundation for new staff of China Medical University, China (Grant No. XZR20160031) and Science Foundation for new staff of China Medical University, China (Grant No. XZR20160030). Author Contributions: Conceived of and designed the experiments: L.W., T.C., M.Z. Performed the experiments: H.W., Q.D., Y.W., C.M. Analysed the data: L.W., Y.W., C.S. Contributed reagents/materials/analysis tools: H.W., Q.D., C.M. Wrote the paper: H.W., Q.D., X.X., M.Z. Edited the paper: X.X., T.C., C.S. Conﬂicts of Interest: The authors declare no conﬂict of interest. Molecules 2017, 22, 1215 8 of 9

Abbreviations The following abbreviations are used in this manuscript:

MDPI Multidisciplinary Digital Publishing Institute DOAJ Directory of open access journals TLA Three letter acronym cDNA Complementary DNA TRH Thyrotropin-releasing hormone RP-HPLC Reverse-phase high performance liquid chromatography BLAST Basic Local Alignment Search Tool NCBI National Centre for Biotechnological Information MALDI-TOF Matrix-assisted laser desorption/ionisation, time-of-ﬂight MS mass spectrometry MS/MS Tandem mass spectrometry SEM Standard error of the mean TFA Triﬂuoracetic acid PS Peptide synthesiser LC/MS/MS Liquid chromatography coupled tandem mass spectrometry

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Sample Availability: Samples of the compounds are available from the authors.

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